Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in Western countries and smoking, diabetes, and pancreatitis are risk factors. The prognosis of this cancer is extremely poor due to its resistance to available therapies and extensive metastasis. New strategies to combat this deadly disease are thus urgently needed and are among priority areas of research identified by the National Cancer Institute. The long-term goal of this project is to develop novel and effective strategies for the treatment and prevention of PDAC. The current project takes advantage of our discovery that beta-adrenoreceptors regulate the growth of human PDAC cell lines and their cells of origin, pancreatic duct epithelial cells. Stimulation of these G- protein coupled receptors by agonists induced signaling via adenylyl cyclase=>cAMP=>PKA=>CREB and transactivated the EGF pathway in a PKA-dependent manner. Isoroterenol additionally stimulated cell migration and had strong anti-apoptotic effects as evidenced by suppression of starvation-induced cleaved casapse 3. GABA and baclophen had strong inhibiting effects on all these responses and additionally reduced proliferation, migration of untreated cells. In support of these in vitro findings, nicotine-induced increase in systemic stress neurotransmitters adrenaline and noradrenaline strongly stimulated the growth of PDAC xenografts, induced p-CREB and p-ERK1/2 in xenograft cells while suppressing the GABA synthesizing enzyme GAD65 and GABA. Treatment of the mice with GABA completely blocked xenograft growth while returning levels of p-CREB, p-ERK1/2, GAD and GABA to normal levels. These data suggest the GABAB receptor as a novel drug target for the treatment and prevention of PDAC. To test this hypothesis we propose four specific aims: Specific aim 1: To evaluate the anti-tumorigenic effects of GABA and the GABAB receptor agonist baclophen on early and advanced stages of PDAC xenograft development in nude mice in the presence and absence of stress neurotransmitter stimulation in response to nicotine and NNK. Specific aim 2: To test the hypothesis that the observed suppression of GAD65 and GABA in nicotine and NNK-treated PDAC xenografts is caused by gene promoter hypermethylation of GAD65, that GABA reverses these effects and to verify these mechanism of gene silencing and reversal in vitro in pancreatic duct epithelial cells. Specific aim 3: To test the hypothesis that the antitumorigenic effects of gefitinib and gemcitabine on PDAC cells are reduced in the presence of stress neurotransmitter stimulation in vitro and in PDAC xenografts and that combination treatments with either agent plus GABA or baclophen improves their effectiveness. Specific aim 4: To assess the effects of the beta-blocker propranolol or the COX-2 inhibitor celecoxib in vitro and in PDAC xenografts with and without stimulation by stress neurotransmitters, and to compare their effectiveness to that of GABA and baclophen. Data to be generated by this project may lead to the successful prevention and treatment of smoking- associated PDAC in a marker-guided fashion with GABA-ergic agents and generate a better understanding of the complex mechanisms of action of stimulating and inhibiting neurotransmitters in the regulation of PDAC. Since GABA and baclophen are already approved for the treatment of non-cancerous conditions in humans, results of this research can be rapidly translated into clinical trials. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is the fourth leading cause of cancer death with mortality near 100% within one year of diagnosis because it does not respond to existing therapies and metastasizes extensively. New strategies to combat this deadly disease are thus urgently needed. Data presented in this project along with known biological effects of risk factors for pancreatic cancer (smoking, diabetes, pancreatitis) suggest that hyperactive cAMP-dependent signaling caused by an increase in the stress neurotransmitters adrenaline and noradrenaline stimulates pancreatic cancer development and progression while at the same time the pancreatic production of gamma-aminobutyric acid (GABA), which normally controls the activity of these receptors via activation of the inhibitory GABAB receptor, is reduced. Based on this novel concept, we propose the GABAB receptor as a novel drug target for the therapy and prevention of pancreatic cancer. In vitro and in vivo studies proposed under this project will provide a preclinical basis for the use of GABA-ergic agents for the effective therapy and prevention of pancreatic cancer.